Binary Patterning Research Articles

Assessment regimes, data, gender haunting, and health education

ABSTRACT Most secondary (high) schools in a broad range of jurisdictions internationally engage in various forms of high stakes, standardized assessment and related qualifications. In this paper, we interrogate how educational achievement regimes – especially via the reporting of curriculum and assessment ‘data’ – continue to mobilize particular gender norms. Drawing on Derrida’s notion of haunting we explore how such regimes impose and reinscribe stable and binary gendered patterning and create what Barad has named ‘entangled relationalities of inheritance’ [https://doi.org/10.3366/drt.2010.0206,] despite young people (and many schools) moving towards greater recognition of non-binary genders. Drawing on assessment data from Aotearoa New Zealand, we look at both generalized reporting of educational achievement data along the lines of ‘male’ and ‘female’ and on reporting of a single (historically gendered) curriculum subject – health education. We argue that such systems are ‘haunted’ by stable gender categorizations and hierarchies and we ask what this means for the reporting of educational assessment data and the erasure of identities that don’t align with the binary.

Simulation study of three-dimensional grayscale ice lithography on amorphous solid water for blazed gratings

Electron beam lithography (EBL) on amorphous solid water (ASW), termed as ice lithography (IL), has demonstrated promising capability in pattern transfer with unique advantages such as reduced proximity effect. So far, ice lithography for binary patterning has been proved a great success, however, application for three-dimensional (3D) profiling in nanoscale has still not been addressed to the best of our knowledge. This paper reports, for the first time, our progress in simulating study of three-dimensional ice lithography on ASW for linear blazed gratings, aiming to overcome the difficulty in replicating high quality blazed gratings with high diffraction efficiency. Systematic simulation of grayscale ice lithography for 3-D blazed grating templates with desired surface quality as the task was carried out, using Monte Carlo algorithm based on the measured contrast curves of ASW. For comparison, grayscale electron beam lithography on PMMA was also performed. The resultant profiles of blazed wavelengths around 1550 nm by grayscale IL show less flaws and higher diffraction efficiencies than by EBL. The successful simulation of 3D grayscale IL provides us with instructive guide for the fabrication of 3D nanostructures as a whole through the grayscale ice lithography on ASW.

Detour-phased perovskite ultrathin planar lens using direct femtosecond laser writing

Perovskite-enabled optical devices have drawn intensive interest and have been considered promising candidates for integrated optoelectronic systems. As one of the important photonic functions, optical phase modulation previously was demonstrated with perovskite substrate and complex refractive index engineering with laser scribing. Here we report on the new scheme of achieving efficient phase modulation by combining detour phase design with 40 nm ultrathin perovskite films composed of nanosized crystalline particles. Phase modulation was realized by binary amplitude patterning, which significantly simplifies the fabrication process. Perovskite nanocrystal films exhibit significantly weak ion migration effects under femtosecond laser writing, resulting in smooth edges along the laser ablated area and high diffractive optical quality. Fabrication of a detour-phased perovskite ultrathin planar lens with a diameter of 150 μm using femtosecond laser scribing was experimentally demonstrated. A high-performance 3D focus was observed, and the fabrication showed a high tolerance with different laser writing powers. Furthermore, the high-quality imaging capability of perovskite ultrathin planar lenses with a suppressed background was also demonstrated.

Single, binary and successive patterning of charged nanoparticles by electrophoretic deposition

Deposition of nanoparticles on a substrate in a controlled manner leads to the formation of multifunctional surfaces and therefore devices. Electrostatic forces can be utilized to manipulate different types of materials such as magnetic, insulating, conducting, semiconducting, organic and inorganic, without altering the chemistry of the surface. However, simultaneous and successive electrophoretic deposition (EPD) methods are not fully utilized for nanoparticles with different characteristics. In this work, electrostatic forces are applied to direct and position charged nanoparticles suspended in aqueous dispersions on desired areas of the surface. Assemblies of particles are obtained by electrostatic attraction generated by gold electrodes of sizes from 500 nm to 50 µm that are fabricated by thermal evaporation. Different types of charged nanoparticles were simultaneously attracted towards different locations of the surface by means of EPD; as a result, alternating nanoparticle patterns and particle deposition on the same designated areas for forming composite areas are obtained. Assemblies formed from positively charged silver nanoparticles and negatively charged fluorescent latex and silica nanoparticles are demonstrated. The position of metallic-, polymeric- and inorganic-based nanoparticles is controlled by the design of electrode geometry.

Modeling binary and graded cone cell fate patterning in the mouse retina

Nervous systems are incredibly diverse, with myriad neuronal subtypes defined by gene expression. How binary and graded fate characteristics are patterned across tissues is poorly understood. Expression of opsin photopigments in the cone photoreceptors of the mouse retina provides an excellent model to address this question. Individual cones express S-opsin only, M-opsin only, or both S-opsin and M-opsin. These cell populations are patterned along the dorsal-ventral axis, with greater M-opsin expression in the dorsal region and greater S-opsin expression in the ventral region. Thyroid hormone signaling plays a critical role in activating M-opsin and repressing S-opsin. Here, we developed an image analysis approach to identify individual cone cells and evaluate their opsin expression from immunofluorescence imaging tiles spanning roughly 6 mm along the D-V axis of the mouse retina. From analyzing the opsin expression of ~250,000 cells, we found that cones make a binary decision between S-opsin only and co-expression competent fates. Co-expression competent cells express graded levels of S- and M-opsins, depending nonlinearly on their position in the dorsal-ventral axis. M- and S-opsin expression display differential, inverse patterns. Using these single-cell data, we developed a quantitative, probabilistic model of cone cell decisions in the retinal tissue based on thyroid hormone signaling activity. The model recovers the probability distribution for cone fate patterning in the mouse retina and describes a minimal set of interactions that are necessary to reproduce the observed cell fates. Our study provides a paradigm describing how differential responses to regulatory inputs generate complex patterns of binary and graded cell fates.

Sequential binary protein patterning on surface domains of thermo-responsive polymer blends cast by horizontal-dipping

We characterize an approach enabling dual protein positioning over broad polymer areas based on subsequent selective adsorption of two fluorescently labelled lectins, Concanavalin A (Con A) and Lentil Lectin (LcH), on self-assembled gradient patterns of thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) and polystyrene (PS) polymers blend, prepared by horizontal dipping technique. The film morphologies of gradient samples prior dual selective protein adsorption are mapped with scanning microscopy (AFM) and secondary ion mass spectrometry (ToF-SIMS), whereas adsorbed proteins are imaged with fluorescence microscope. ToF-SIMS analysis reveals surface composition consisting of PNIPAM-rich domains in PS-rich matrix. The two-step protein adsorption experiment results in selective adsorption of Con A and LcH to PNIPAM- and PS-rich phases, respectively. Integral geometry approach is used to compare quantitatively morphology of polymer patterns varied in domain size due to horizontal dipping casting. Minkowski measures are also used to compare quantitatively fluorescence micrographs of protein patches with SIMS images of original isotropic polymer patterns. It confirms that PNIPAM domains size increases with increasing speed. Further, Minkowski analysis unveiled that adsorbed proteins cover about 60–70% of polymer surface. What is more fluorescence micrographs acknowledge both no lectins contamination and no adsorption to interphase areas. Additionally, protein displacement effect is observed.

BMP and FGF signaling interact to pattern mesoderm by controlling basic helix-loop-helix transcription factor activity.

The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF-mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer.

MARS: A protein family involved in the formation of vertical skeletal elements.

Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements.

Interplay between Conformational Heterogeneity and Hydration in the Folding Landscape of a Designed Three-Helix Bundle.

Water is known to play a critical role in protein folding and stability. Here we develop and employ a coarse-grained (CG) model to directly explore the role of water in shaping the conformational landscape explored during protein folding. For this purpose, we simulate a designed sequence with binary patterning of neutral and hydrophobic residues, which is capable of folding to a three-helix bundle in explicit water. We find two folded states of this sequence, with rotation of the helices occurring to trade between hydrophobic packing and water expulsion from the core. This work provides insight into the role of water and hydrophobicity in generating competing folded states for a protein.

Exploring productive sequence space in directed evolution using binary patterning versus conventional mutagenesis strategies

Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening. In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept of binary patterning, with and without additional hydrophobic amino acids. It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the production of both (R,R)- and (S,S)-diols (80–97 % ee), but again the introduction of polar amino acids occurs rarely. Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols are not always accessible.

De Novo Construction of Redox Active Proteins.

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways.

Linking metrics of landscape pattern to hydrological process in a lotic wetland

Strong reciprocal interactions exist between landscape patterns and ecological processes. In wetlands, hydrology is the dominant abiotic driver of ecological processes and both controls, and is controlled, by vegetation presence and patterning. We focus on binary patterning in the Everglades ridge-slough landscape, where longitudinally connected flow, principally in sloughs, is integral to landscape function. Patterning controls discharge competence in this low-gradient peatland, with important feedbacks on hydroperiod and thus peat accretion and patch transitions. To quantitatively predict pattern effects on hydrologic connectivity and thus hydroperiod. We evaluated three pattern metrics that vary in their hydrologic specificity. (1) Landscape discharge competence considers elongation and patch-type density that capture geostatistical landscape features. (2) Directional connectivity index (DCI) extracts both flow path and direction based on graph theory. (3) Least flow cost (LFC) is based on a global spatial distance algorithm strongly analogous to landscape water routing, where ridges have higher flow cost than sloughs because of their elevation and vegetation structure. Metrics were evaluated in comparison to hydroperiod estimated using a numerically intensive hydrologic model for synthetic landscapes. Fitted relationships between metrics and hydroperiod for synthetic landscapes were extrapolated to contemporary and historical maps to explore hydroperiod trends in space and time. Both LFC and DCI were excellent predictors of hydroperiod and useful for diagnosing how the modern landscape has reorganized in response to modified hydrology. Metric simplicity and performance indicates potential to provide hydrologically explicit, computationally simple, and spatially independent predictions of landscape hydrology, and thus effectively measure of restoration performance.

Binary gene expression patterning of the molt cycle: the case of chitin metabolism.

In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish Cherax quadricarinatus, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes.

High-quality combinatorial protein libraries using the binary patterning approach.

Protein combinatorial libraries have become a platform technology for exploring protein sequence space for novel molecules for use in research, synthetic biology, biotechnology, and medicine. To expedite the isolation of proteins with novel/desired functions using screens and selections, high-quality approaches that generate protein libraries rich in folded and soluble structures are desirable for this goal. The binary patterning approach is a protein library design method that incorporates elements of both rational design and combinatorial diversity to specify the arrangement of polar and nonpolar amino acid residues in the context of a desired, folded tertiary structure template. An overview of the considerations necessary to design and construct binary patterned libraries of de novo and natural proteins is presented.

Directed evolution of the peroxidase activity of a de novo-designed protein

Collections of de novo-designed proteins provide a unique opportunity to probe the functional potential of sequences that are stably folded, but were neither explicitly designed nor evolutionarily selected to perform any particular type of activity. A combinatorial library of folded proteins was designed previously using a strategy that exploits the binary patterning of polar and non-polar amino acids to drive sequences to fold into four-helix bundles. Although these novel four-helix bundles were not explicitly designed for function, previous characterization of several hundred arbitrarily chosen sequences showed that many of them bound the heme cofactor, and several of these novel heme proteins catalyzed peroxidase activity at levels substantially above background. Here, we show that these moderately active de novo heme proteins can serve as non-natural starting points for laboratory-based evolution: Random mutagenesis followed by color-based screening of a relatively small number (hundreds or thousands) of variants yielded novel sequences with improved peroxidase activity. Biochemical characterization of the purified proteins showed that the evolved variants were nearly 3-fold more active than the parental sequence. These results demonstrate that de novo-designed proteins can be utilized as a novel feedstock for the evolution of enzyme activity.

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

Colloidally Templated Two-Dimensional Conducting Polymer Arrays and SAMs: Binary Composition Patterning and Chemistry

A facile approach and strategy toward binary-composition, two-dimensional (2D) patterned surfaces of conducting polymer periodic arrays, together with thiol self-assembled monolayers (SAMs) is described. The method involved a Langmuir-Blodgett (LB)-like deposition of latex microsphere particles, electropolymerization via cyclic voltammetric (CV) techniques, and self-assembly of an amphiphile. The LB-like technique enabled the monolayer deposition of different sizes of polystyrene (PS) particles in hexagonal packing arrangement on planar substrates. Combining the LB-like method with CV electropolymerization is advantageous because it provides deposition control of a polymer interconnected network, controlled composition ratio of polymer and SAMs, and control of 2D size and spacing of the spherical void pattern. Electrochemical-quartz crystal microbalance (EC-QCM) in situ monitoring of the film deposition quantified a constant and linear growth rate, with varying viscoelastic behavior of the conducting polymer adsorption on planar and PS-templated substrates. The dual-patterned surface provided a good imaging contrast as observed by atomic force microscopy (AFM). Complementary analyses such as X-ray photoelectron spectroscopy (XPS), attenuated total internal reflection infrared (ATR IR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, and static contact angle measurements were used to characterize the formation of the patterned surface. The versatility of the method enables the potential for making various types of quantitative binary compositions and patterned surfaces using different combinations of conducting polymer or functional SAMs, which can be extended in the future to polymer brushes and layer-by-layer assembly of various materials.

De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth

A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >106 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains – ΔserB, ΔgltA, ΔilvA, and Δfes – were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.

Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

Neutrality and evolvability of designed protein sequences

The effect of foldability on protein's evolvability is analyzed by a two-prong approach consisting of a self-consistent mean-field theory and Monte Carlo simulations. Theory and simulation models representing protein sequences with binary patterning of amino acid residues compatible with a particular foldability criteria are used. This generalized foldability criterion is derived using the high temperature cumulant expansion approximating the free energy of folding. The effect of cumulative point mutations on these designed proteins is studied under neutral condition. The robustness, protein's ability to tolerate random point mutations is determined with a selective pressure of stability (ΔΔG) for the theory designed sequences, which are found to be more robust than that of Monte Carlo and mean-field-biased Monte Carlo generated sequences. The results show that this foldability criterion selects viable protein sequences more effectively compared to the Monte Carlo method, which has a marked effect on how the selective pressure shapes the evolutionary sequence space. These observations may impact de novo sequence design and its applications in protein engineering.